Abstract
The mechanism by which Z-338, a novel gastroprokinetic agent, stimulates gastric motility was studied in relation to muscarinic receptors in the guinea pig. Z-338 (3–30 μM) enhanced electrically stimulated contractions and the release of acetylcholine (ACh) that was tetrodotoxin sensitive and extracellular Ca2+ dependent, in gastric strips. Membrane-binding assay revealed that Z-338 possessed binding affinity for muscarinic M1and M2, but not M3 receptors. InXenopus oocytes expressing M1 and M2 muscarinic receptors, Z-338 did not produce any response, but inhibited ACh-induced outward currents, thereby indicating that Z-338 acts on the M1 and M2muscarinic receptors as an antagonist. The M1 receptor antagonist pirenzepine (0.5 μM) and M2 receptor antagonist AF-DX 116 (1 μM) also enhanced electrically stimulated release of ACh. These results indicate that Z-338 facilitates ACh release from cholinergic nerve terminals by blocking muscarinic M1 and M2 autoreceptors, which regulate the release of ACh.
Z-338 [N-(N′,N′-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxy-benzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate] (Fig.1) is a newly synthesized gastroprokinetic agent that enhances gastrointestinal motility in conscious dogs and gastric emptying in rats and dogs (Ueki et al., 1998). In an in vitro study, Z-338 inhibited the activity of acetylcholinesterase derived from human erythrocyte membranes and produced a contraction of antrum preparations from guinea pig stomach (Kurimoto et al., 1998). The most widely used gastrointestinal prokinetic benzamides interact with 5-hydroxytryptamine (5-HT) receptors, especially the 5-HT4 receptor subtype to facilitate acetylcholine (ACh) release from enteric neurons (Taniyama et al., 1991). Z-338 does not possess binding affinity for 5-HT receptors (Kurimoto et al., 1998), and the mechanism underlying its gastroprokinetic action has not been elucidated. Thus, this study was attempted to determine whether Z-338 stimulates the release of ACh from strips isolated from the guinea pig stomach and interacts with muscarinic receptors with membrane receptor-binding assay andXenopus oocytes heterologously expressing cloned receptors.
Materials and Methods
Preparation of Stomach Strips.
Male Hartley guinea pigs weighing 300 to 500 g (Kyudo Inc., Kumamoto, Japan) were decapitated. The whole stomach was dissected out and placed in Krebs-Henseleit solution (118 mM NaCl, 4.8 mM KCl, 1.18 mM KH2PO4, 1.19 mM MgSO4, 2.5 mM CaCl2 2.5, 25 mM NaHCO3, and 11 mM d-glucose). The antrum and corpus of the stomach were immediately cut into circular strips ∼10 × 2 mm. The mucosa was rapidly removed from the tissue and the muscle layers and intramural plexus were left intact.
Measurement of Mechanical Activity.
The strips were mounted in a superfusion apparatus with resting tension of 0.5 g and superfused at a constant rate of 1.2 ml/min with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2 at 37°C for 80 min. The tension was kept constant by readjustment during the equilibration period. The strips were stimulated with two parallel platinum electrodes for 2 min of 1-ms duration, 10-V intensity, and a frequency of 1 Hz successively three times (S1, S2, S3) at 30-min intervals. When the effect of Z-338 on electrically stimulated contractions was evaluated, Z-338 was applied to the superfusion solution 10 min before the third stimulation (S3). The contractile activity was analyzed quantitatively by measuring the area under the contractile waves with Flexi Trace (Tree Star, Inc., San Carlos, CA). The ratio of S3/S2 calculated from the S2 and S3 without Z-338 was used as a control, and the effect of Z-338 on the electrically stimulated contraction was evaluated by the ratio of S3/S2 calculated from the S3 in the presence of Z-338.
Measurements of Tritium Outflow.
The methods of incubation and superfusion were as described by Kusunoki et al. (1985) and Takeda et al. (1991). The strips were incubated with [3H]choline (2997 GBq/mmol; DuPont-NEN, Boston, MA) at a final concentration of 90 nM in oxygenated Krebs-Henseleit solution at 37°C for 60 min. At the end of the labeling period, the strips were mounted in a superfusion apparatus and washed out by superfusion with Krebs-Henseleit solution gassed with 95% O2 and 5% CO2 at a constant rate of 0.6 ml/min for 60 min at 37°C. Hemicholinium-3 (10 μM) was present in the superfusion solution to prevent the uptake of [3H]choline. Two parallel platinum electrodes were used to stimulate intramural nerves. The strips were stimulated electrically at parameters of 1-ms duration, 15-V intensity, and a frequency of 5 Hz for 30 s. The strip was stimulated successively four times (S1, S2, S3, and S4) at 34-min intervals, at 6 min (S1), 40 min (S2), 74 min (S3), and 108 min (S4) after the end of washout. The superfusate was collected every 2 min and the radioactivity of the sample was determined by counting in a liquid scintillation spectrometer (Packard Instrument Co., Downers Grove, IL). The radioactivity of the tissue dissolved in Soluene-350 (Packard Instrument Co.) at the end of the release experiment was measured in a liquid scintillation spectrometer.
The validity of assuming total tritium as a measure of [3H]ACh release under the present experimental conditions has been documented in our previous studies (Kusunoki et al., 1985; Takeda et al., 1991). The outflow of tritium was represented as the fractional rate obtained by dividing the amount of tritium in the superfusate by the respective amount of tritium in the tissue. The tritium content of the tissue in each period was calculated by adding cumulatively the amount of each fractional tritium outflow, to the tritium content of the tissue at the end of the experiment. From each of the outflow curves obtained by plotting the fractional outflow of tritium against time, the outflow of tritium evoked by stimulation in each condition was calculated from the difference between the peak tritium outflow and the basal outflow. The ratio of S3/S2 calculated from the S2 and S3 without substances was used as a control, and the effects of substances on the electrically evoked outflow were evaluated by the ratio of S3/S2 calculated from the S3 in the presence of substances. When the effect of Z-338 on the electrically stimulated outflow of tritium in the presence of physostigmine was examined, physostigmine at 0.1 μM was contained in the superfusion solution throughout the experiment. Calcium free Krebs-Henseleit solution was prepared by replacing CaCl2 with MgCl2 and contained 1 mM EGTA.
Receptor-Binding Assay.
The methods of receptor-binding assays for muscarinic M1, M2, and M3 were carried out according to the methods of Wang et al. (1987; M1muscarinic receptor) and Delmendo et al. (1989; M2 and M3 muscarinic receptors), respectively. The cerebral cortex, heart, and submaxillary gland were dissected from male Sprague-Dawley rats (250–350 g). The tissues were homogenized separately in 40 volumes (cerebral cortex), 20 volumes (heart), and 30 volumes (submaxillary gland) of ice-cold buffer (50 mM sodium/potassium phosphate buffer, pH 7.4, for the cortex and 50 mM Tris-HCl buffer with 5 mM EDTA, pH 7.4, for the heart and submaxillary gland) with an Ultra-Turrax homogenizer (Janke and Kunkel, Staufen, Germany) for 30 s, three times (cerebral cortex), and 20 sec, two times (heart and submaxillary gland). The homogenates of heart and submaxillary gland were passed through a double layer of cheesecloth. The homogenates were centrifuged at 1,000g for 5 min (cerebral cortex) and 500g for 10 min (heart and submaxillary gland) at 4°C, and then the supernatants were centrifuged at 40,000g for 20 min (cerebral cortex) and 30,000g for 15 min (heart and submaxillary gland). The pellets were washed by resuspension in 30 volumes of ice-cold buffer and subsequently centrifuged at 40,000g for 20 min (cerebral cortex) and 30,000g for 15 min (heart and submaxillary gland). The final pellets were resuspended in 40 ml (cerebral cortex) and 30 ml (heart and submaxillary gland) of ice-cold buffer and frozen at −80°C until assay.
The membrane preparations from cerebral cortex, heart, and submaxillary gland were exposed to 1 nM [3H]pirenzepine (2752.8 GBq/mmol; DuPont-NEN; M1 muscarinic receptor) and 0.2 nMN- [3H]methylscopolamine (NMS, 3180.0 GBq/mmol; DuPont-NEN; M2 muscarinic receptor for heart and M3 muscarinic receptor for submaxillary gland), respectively. Membranes (0.2 mg of protein/tube) were incubated with tritium ligands in assay buffer (50 mM sodium/potassium phosphate buffer, pH 7.4, for cerebral cortex, 50 mM Tris-HCl buffer containing 5 mM EDTA and 5 mM MgCl2, pH 7.4, for heart and submaxillary gland) and various concentrations of Z-338 and other positive control substances. Total volume of the incubation mixture was 1 ml. Tubes were incubated for 60 min (cerebral cortex) and 90 min (heart and submaxillary gland) at 25°C. After incubation, the labeled membranes were harvested and washed four times with 3 ml of ice-cold buffer by rapid vacuum filtration through Whatman GF/B filters that had been presoaked in 0.1% polyethyleneimine. The radioactivity on the filters was measured with a liquid scintillation spectrometer. Specific binding was defined as the excess over blank in the presence of 1 μM atropine for each receptor. The IC50 value of the specific binding of the tritium ligand was computed by the logit-log analysis and the inhibition affinity constant (Ki) was obtained according to the method of Cheng and Prusoff (1973). Saturation-binding data were converted to a Scatchard plot, from which the affinity constantsKd andBmax were determined as −1/slope andx-axis intercept, respectively.
Expression of Muscarinic M1 and M2Receptors in Xenopus oocytes.
The cRNAs for rat M1 muscarinic receptor and human M2 muscarinic receptor were synthesized in vitro with T7 polymerase with Ambion's MEGAscrip kits (Austin, TX) from linearized cDNAs with HindIII (Matsumoto et al., 1998;Uezono et al., 1998). Xenopus were anesthetized by hypothermia. Small incisions were made on Xenopus's abdomen, and portions of the ovary removed (Dascal et al., 1985). Stage V–VI (Dascal, 1987) Xenopus oocytes were isolated and deffoliculated by gently shaking them at room temperature (21–23°C) for 60 min in Ca2+-free solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, and 10 mM HEPES, pH 7.4) containing 0.5 mg/ml collagenase (Yakult, Tokyo, Japan). cRNAs (5 ng) for M1 or M2 receptor were injected into the oocytes with a Picospritzer II (General Valve Co., Fairfield, NJ); oocytes were then incubated at 19°C in modified Barth's solution (88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, and 10 mM HEPES, pH 7.4) containing 2.5 mM sodium pyruvate and 20 mg/ml gentamycin for 2 to 4 days. The oocytes were incubated in modified Barth's solution that did not contain gentamycin >3 h before electrophysiological study. For the experiment on the M2 receptor, 5 ng of cRNA for the G protein-gated inward rectifying K+ channel (GIRK1) was injected into the oocytes together with cRNA for M2 receptor. GIRK1 was synthesized in vitro with T3 polymerase with Ambion's MEGAscrip kits from linearized cDNA for rat GIRK1 with SalI (Uezono et al., 1998). The Ca2+-free solution and modified Barth's solution were sterilized before use.
Electrophysiological measurements were made between 2 and 4 days after cRNA injection with a two-microelectrode voltage clamp amplifier (TEV-200; Dagan Instruments, Minneapolis, MN). The oocyte membranes were clamped at −60 mV and continuously superfused with bath solution containing 80 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES, pH 7.4. The bath had a volume of 150 μl and the flow rate was 2 ml/min. Each concentration of substance was dissolved in bath solution and superfused at the same flow rate for 30 s, unless otherwise indicated. The effects of Z-338 on the ACh (1 μM)-induced inward current were determined from the peak amplitude of the ACh-induced inward current 30 s after treatment with Z-338. For determination of the effect of Z-338 on the M2 receptor, the bath solution was exchanged for high K+ solution (2 mM NaCl, 96 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM HEPES, pH 7.4). The solution in the bath was replaced completely within 20 s. The reversal potential of induced currents was measured using a ramp method with a multifunction synthesizer (NF, Tokyo, Japan). The currents induced by the ramp waves were fed into a personal computer (NEC, Tokyo, Japan) and analyzed.
Drugs and Chemicals.
The drugs and chemicals used were as follows: Z-338 and AF-DX 116 [11,2-(diethylamino)methyl-1-piperidinyl-acetyl-5,11-dihydro-6H-pyrido-2,3b-1,4-benzodiazepine-6-one] (a gift from Zeria Pharmaceutical Co., Ltd., Tokyo, Japan); [3H]choline chloride, [3H]pirenzepine, and [3H]NMS (DuPont-NEN); acetylcholine chloride, hemicholinium-3, and pirenzepine dihydrochloride (Sigma, St Louis, MO); tetrodotoxin (TTX; Wako Pure Chemical, Osaka, Japan); Soluene-350 (Packard Instrument Co.); and EGTA (Nacarai Tesque, Kyoto, Japan). Drugs were dissolved in distilled water, then diluted with buffer to the required concentration.
Statistical Analysis.
All data are represented as the mean ± S.E. A statistical analysis between the control and substance-treated group was made with Dunnett's test or the Mann-Whitney U test (MUSCOT Statistical Analysis Program; Yukms Co., Ltd., Tokyo, Japan). A P value <.05 was considered statistically significant.
Results
Effect of Z-338 on Electrically Stimulated Contractions of Strips of Stomach.
Electrical transmural stimulation (1 Hz, 10 V, 1 ms) for 2 min caused contraction of the strips. This contraction was prevented by application of 1 μM atropine or 0.3 μM TTX to the superfusion solution 10 min before the stimulation. When Z-338 at concentrations of 3 to 100 μM was applied to the superfusion solution 10 min before the electrical stimulation, the contraction was enhanced in a concentration-dependent manner (Fig.2).
Effect of Z-338 on Electrically Stimulated Outflow of Tritium.
The outflow of tritium was studied in the presence of TTX, or after calcium had been omitted from the Krebs-Henseleit solution. TTX (0.3 μM, pretreatment for 10 min) and omission of calcium (pretreatment for 20 min) completely and reversibly depressed the electrically stimulated outflow of tritium, as noted by Kusunoki et al. (1985) andTakeda et al. (1991), thereby indicating that the outflow of tritium is neuronal in origin. Z-338 (30 μM) significantly enhanced the electrically stimulated outflow of tritium (Fig.3).
The enhancing effect of 10 μM Z-338 on the electrically stimulated outflow of tritium was examined in strips in which the cholinesterase activity was inhibited by physostigmine. The superfusion solution used throughout the experiment contained 0.1 μM physostigmine. In the control experiment, the ratio of S3 (in the absence of Z-338)/S2 (in the absence of Z-338) was 1.02 ± 0.06 (n = 6). When the effect of Z-338 was examined, the ratio of S3 (in the presence of Z-338)/S2 (in the absence of Z-338) was 1.73 ± 0.11 (n = 6). Z-338 at 10−5 M significantly enhanced the electrically stimulated outflow of tritium, and this enhancement was greater in the presence of physostigmine than in its absence.
Effects of Pirenzepine and AF-DX 116 on Electrically Stimulated Outflow of Tritium.
Pretreatment with pirenzepine at 0.5 μM for 10 min enhanced the electrically stimulated outflow of tritium (Fig.4). Pretreatment with AF-DX 116 at concentrations of 1 and 10 μM also enhanced the electrically stimulated outflow of tritium (Fig. 4).
Receptor-Binding Assay.
Z-338 displaced the specific binding of [3H]pirenzepine to rat cortex membrane (M1 receptor) and [3H]NMS to rat heart membrane (M2 receptor), but did not displace the specific binding of [3H]NMS to rat submaxillary gland membrane (M3 receptor; Fig.5). TheKi values of M1and M2 receptors were 8.4 and 9.4 μM, respectively.
Effect of Z-338 on Muscarinic M1 and M2Receptors Expressed in Xenopus Oocytes.
In oocytes expressing the M1 receptor, ACh at 1 μM produced a Ca2+-activated Cl− current. Reapplication of the same concentration of ACh 30 min later produced a similar magnitude of current (data not shown). Z-338 did not produce any currents, whereas pretreatment with Z-338 for 30 s attenuated the ACh-induced Ca2+-activated Cl− current in oocytes expressing the M1 receptors, in a dose-dependent manner (Fig. 6A).
ACh at 1 μM produced an inward K+ current through GIRK1 in the oocytes expressing the M2receptor. Z-338 did not produce any currents, but as shown in Fig. 6B, Z-388 also attenuated the ACh-induced inward K+current, in a concentration-dependent manner.
Discussion
Z-338 enhanced the electrically stimulated contractions of strips isolated from guinea pig stomach, in a concentration-dependent manner. The contractions were sensitive to TTX and atropine, therefore the enhancement by Z-338 is probably due to an increase in ACh release. This concept is supported by the finding that Z-338 enhanced the electrically stimulated outflow of tritium. The electrically stimulated outflow of tritium was TTX sensitive and extracellular Ca2+ dependent, and the validity of assuming total tritium as a measure of [3H]ACh release has been documented in our previous studies with stomach strips (Kusunoki et al., 1985; Takeda et al., 1991).
Z-338 does not possess binding affinity for the serotonin 5-HT4 receptor (Kurimoto et al., 1998); however, the receptor-binding assay revealed that Z-338 bound to muscarinic M1 and M2 receptors but not to the muscarinic M3 receptor. Whether Z-338 is an agonist or antagonist for muscarinic M1 and M2 receptors was examined in Xenopusoocytes expressing one or the other of these receptors. Z-338 alone did not produce any currents in the oocytes expressing either M1 or M2 receptors, but inhibited the ACh-induced inward currents mediated by stimulation of M1 and M2 receptors. Thus, Z-338 acts as an antagonist at M1 and M2 receptors.
In gastrointestinal tissues, M1, M2, and M3 receptors are reported to be present in the neurons and/or smooth muscle cells. The muscarinic receptors present in the neurons appear to be located on the cholinergic nerve terminals and to operate as autoreceptors. The release of ACh from cholinergic nerves is modulated by a negative feedback mechanism that is triggered by stimulation of presynaptic muscarinic receptors. Such an autoinhibition of ACh release has been detected in many tissues (Starke et al. 1989). In the enteric nervous system, previous studies have indicated that the release of ACh from guinea pig myenteric and submucous plexus neurons is inhibited by the presynaptic M1 receptor (Kawashima et al., 1988;Schwörer and Kilbinger, 1988; Kilbinger et al., 1993; Dietrich and Kilbinger, 1995) and M2 receptor in rat antral mucosal/submucosal neurons (Ren and Harty, 1994). The effects of pirenzepine (M1 antagonist) and AF-DX 116 (M2 antagonist) on release of ACh were examined. These selective muscarinic antagonists enhanced the electrically stimulated release of ACh. Thus, the facilitation of ACh release in the presence of pirenzepine and AF-DX 116 may be attributed to a blockade of the presynaptic M1 and M2 autoreceptors that are activated by ACh released from the nerve terminals. It has been reported that pirenzepine accelerates gastrointestinal transit time (Jaup et al., 1985) and increases the frequency of antral contraction (Stacher et al., 1982) in humans.
Because Z-338 inhibits the activity of cholinesterase (Kurimoto et al., 1998), the enhancing effect of Z-338 on ACh release was examined under the conditions of inhibition of cholinesterase activity by physostigmine. Z-338 enhanced the electrically stimulated release of ACh to a greater extent when physostigmine was present in the superfusion solution. Thus, Z-338 apparently does not increase the apparent outflow of tritium simply by inhibiting acetylcholinesterase. Similarly, Mike (1994) suggested that the effect of atropine on the stimulated release of ACh was much greater in the presence of physostigmine than in its absence. In the guinea pig ileum, scopolamine has been shown to greatly facilitate the stimulated release of ACh in the presence of cholinesterase inhibitor (Kilbinger and Wessler, 1980). Thus, ACh release is clearly enhanced by inhibition of muscarinic autoreceptors with muscarinic antagonists when the cholinesterase activity is inhibited.
Although Z-338 antagonized M1 and M2 receptors it had no effect on the M3 receptor, and had the same effect on the release of ACh as did other antagonists for M1and M2 receptors. Thus, facilitation of ACh release from cholinergic nerve terminals of guinea pig stomach by blockade of presynaptic muscarinic autoreceptors may be one mechanism by which Z-338 facilitates gastric motility. A substance such as Z-338 that is an antagonist for muscarinic autoreceptors, but not for the muscarinic M3 receptor, may soon be available for clinical use as a gastroprokinetic agent possessing a new mechanism of action.
Acknowledgments
We thank Naoki Tuzuike and Kenji Yoshida for excellent technical assistance.
Footnotes
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Send reprint requests to: Kohtaro Taniyama, M.D., Ph.D., Department of Pharmacology, Nagasaki University School of Medicine, Nagasaki 852-8523, Japan. E-mail: taniyama{at}net.nagasaki-u.ac.jp
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↵1 This study was supported by grants from the Ministry of Education, Science, Sports and Culture, Japan.
- Abbreviations:
- Z-338
- N-(N′,N′-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxy-benzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate
- 5-HT
- 5-hydroxytryptamine
- ACh
- acetylcholine
- NMS
- N-methyl scopolamine
- GIRK1
- G protein-gated inward rectifying K+ channel
- AF-DX 116
- 11,2-(diethylamino)methyl-1-piperidinyl-acetyl-5,11-dihydro-6H-pyrido-2,3b-1,4-benzodiazepine-6-one
- TTX
- tetrodotoxin
- Received September 20, 1999.
- Accepted March 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics